DNA, Protein Domains, Huntington’s Disease, and Replication

Posted on Jan 27, 2025 in Biotechnology

Hypothesis on DNA Melting Temperature (Tm) and GC Content

It was hypothesized that DNA with a higher GC content and normal high salt concentrations (Na+/K+) would have a higher Tm. To prove or disprove this hypothesis, researchers took different kinds of animal DNA (stripped of proteins) with varying GC content. The GC content was measured using biochemical methods. The Tm is the temperature at which half of the DNA is single-stranded and the other half is double-stranded. Heat was added to the various DNA samples from 60-100°C, and the Tm was recorded. Synthetic DNA with 100% GC and 100% AT were used as reference points.

The results were graphed with the y-axis representing GC percentage and the x-axis representing Tm. The higher the GC content, the more the graph shifted to the right, indicating a higher Tm. High salt concentrations also contributed to the right shift because high salt content shields the negative charge of DNA, making it more difficult for the DNA strands to unwind. In contrast, low salt concentrations cannot shield the negative charge as effectively, making it easier for the DNA to unwind. Therefore, DNA with high salt concentrations and higher GC content will have higher Tm values than DNA with low salt concentrations and low GC content.

Protein Domains: Independent Folding and Function

It was hypothesized that proteins have domains that fold and function independently. To test this hypothesis, researchers used the well-known src protein, which contains three domains: SH3 (polyproline-binding), SH2 (phosphotyrosine-binding), and SH1 (tyrosine kinase domain). Each domain was isolated and tested.

Reagents/Assay:

  • The SH3 domain was isolated from the rest of the protein by adding two flanking oligo pairs: one for the start at the beginning and one for the stop at the end of the sequence domain.
  • This was then amplified by PCR and added to a plasmid, which was introduced into a bacterial cell to produce the protein domain.
  • The domain was visualized using X-ray diffraction. This process was repeated for the other domains.
  • Another way to test if the SH3 domain folded correctly was by adding polyproline to the plasmid and observing if it would bind to the fully folded SH3 domain.

Results: Proteins have domains that fold and function independently. This was demonstrated by showing that the domains in the src protein fold and function independently of one another, meaning that the primary amino acid sequence is the driving force in protein folding.

Huntington’s Disease: Genetic Testing and Diagnosis

Huntington’s disease (HD) is an autosomal dominant disorder caused by a gain-of-function mutation resulting in CAG trinucleotide repeats in the HTT gene. Normal individuals have 6-35 repeats, while affected individuals have more than 35 repeats. The sequences around the repeats are well-known, so isolating the repeats to predict the genotype is fairly straightforward.

Reagents/Assay:

  • White blood cells (WBCs) are used as the DNA source.
  • The cells are stripped of proteins.
  • The CAG repeat region is isolated using specific oligo pairs and amplified using PCR.
  • The DNA fragments are run on a non-denaturing gel and visualized with ethidium bromide (EtBr) staining under UV light.

Case Study:

Three individuals were tested:

  • Individual 1: 25/25 CAG repeats
  • Individual 2: 25/50 CAG repeats
  • Individual 3: 25/100 CAG repeats

Since HD is a dominant disorder, it is likely that:

  • Individual 1 will not have HD.
  • Individuals 2 and 3 are likely to have HD.
  • Individual 3 will likely have a severe case with early onset.

Important Note: Before informing the patients, 30x next-generation sequencing (NGS) should be conducted to double-check the results and ensure the accuracy of the diagnosis.

DNA Replication: Order of Protein Binding

It was hypothesized that there is a distinct order of protein binding to DNA during replication. Radiolabeled 32P-DNA containing the origin of replication was used, along with different combinations of ORC (origin recognition complex), Cdc1/Cdc6, and helicase. The proteins were also added individually as controls. The results were visualized on a non-denaturing gel with EtBr staining and UV light. The DNA migrates from top to bottom, with the least molecular weight at the bottom.

Results and Conclusions:

  • Without any protein, only 32P-DNA is visible.
  • With ORC, 32P-DNA with bound ORC appears.
  • With ORC and Cdc1/Cdc6, 32P-DNA with bound ORC and Cdc1/Cdc6 appears.
  • With ORC, Cdc1/Cdc6, and helicase, 32P-DNA with all three proteins bound appears.
  • The controls, except for the ORC control, show only 32P-DNA. The ORC control shows 32P-DNA and ORC.

Conclusion: These results demonstrate that proteins involved in DNA replication have an ordered binding pattern. Proteins involved in DNA replication require the protein that was bound before it in the pre-replication complex to bind successfully.